Chemical exposures are important risks for development of hepatocellular carcinoma (HCC). One such chemical, diethylnitrosamine (DENA), is present in food products as well as in industrial and research settings. Further examination of tumors induced by DENA may yield clues to human risk. HCC from seven rhesus macaques exposed to DENA was selected from a tissue archive to examine for evidence of Wnt/β-catenin signaling events, which are frequently associated with HCC. DENA exposure durations ranged from 8 to 207 months, and total accumulated dose ranged from 0.7 to 4.08 mg. Unexposed colony breeder macaques served as controls. Previously unrecognized HCC metastases were discovered in lungs of three macaques. Overexpression of β-catenin and glutamine synthetase was detected by immunohistochemistry in six confirmed primary HCC and all metastatic HCC, which implicated Wnt/β-catenin activation. Concomitant β-catenin gene mutation was detected in one primary HCC; similar findings have been reported in human and rodent HCC. Neither β-catenin mutation nor β-catenin overexpression appeared to influence metastatic potential. Accumulation of intracellular proteins involved in Wnt/β-catenin signaling during HCC oncogenesis in rhesus macaques exposed to DENA appears to include other mechanisms, in addition to mutation of β-catenin gene.